Summary Interleukin-4 is a prototypic immunoregulatory cytokine. It is the central regulator of allergic inflammatory responses, controlling the polarization of naive CD4 T cells to the Th2 phenotype and Ig class switching to IgE. The Cytokine Biology Unit has characterized the signaling mechanisms utilized by the IL-4 receptor. It has shown that activation of the latent transcription factor, Stat 6, controls both TH2 polarization and IgE class switching and that GATA3 and Stat5 play essential roles in the acquisition of IL-4 producing capacity. Unit scientists have recently carried out genome-wide analyses of histone modification and transcription factor binding in each of the various Th lineages to gain insight into the processes underlying Th differentiation. They have recently identified the large number of sites to which GATA3 binds and sites for other transcription factors that bind in close proximity suggesting that these transcription factors act together with GATA3 in mediating transcriptional regulation. This information coupled with the knowledge of the binding sites for STAT5 obtained by our colleague Warren Leonard will provide important insights into the genetic regulation of Th2 differentiation. Comparable studies on the other Th lineages should provide similar information for the differentiation of those cells. Our analysis has already shown that GATA3 may mediate specific repressive functions in lineages other than Th2 cells and thus contribute to shaping the pattern of gene expression in various cell populations. Proteomic studies using mass spectrometry has identified several GATA3 partners of which three appear particularly interesting. They are BCL11b, Ikaros, and E$BP4 (NF-IL-3). Studies are in progress to identify those sites to which both GATA3 and each bind and to determine the consequences of knocking down or knocking out these transcription factors on GATA3 functions. Unit scientists have now been shown that T cell receptor-mediated activation of the erk signaling pathway blocks Th2 differentiation by preventing transcription of GATA3 and desensitizing the IL-2 receptor. Strong TCR-mediated signals stimulate erk phosphorylation, thereby preventing TH2 differentiation and accounting for poor Th2 differentiation at high antigen concentration. Low concentrations of antigen, which activate erk only weakly, are permissive for early IL-4 production and TH2 differentiation. The targets of erk action that mediate suppression of GATA3 transcription are under active study as are the transcription factors that mediate TCR induced GATA3 transcription. We have confirmed that Notch signaling participates in Th2 priming but shown that it does so not by regulating GATA3, as others had proposed, but rather by regulating both early IL-2 production and the sensitivity of the IL-4 receptor to IL-4 by increasing the level Stat-6 phosphorylation to a standard challenge of IL-4. The defect in Th2 priming in mice with defects in the Notch pathway can be rescued by the addition of IL-2, although larger amounts of IL-2 are needed for increasing the Th2 response in the absence of Notch than in its presence. Gfi-1, in its role as a transcriptional repressor, is critical for robust growth to IL-4, since it represses expression of genes such as SOCS1 and SOCS3 that inhibit IL-2 mediated growth. On the other hand, Gfi-1 diminishes growth in response to IL-7 since it mediates TCR- and cytokine-mediated down regulation of the IL-7R alpha chain. These studies, utilizing Gfi-1 conditional knockout mice developed in the Unit, establish that Gfi-1 plays a critical role in lymphocyte homeostasis and that precise regulation of Gfi-1 expression is central to a balanced population of CD4 and CD8 T cells. Further, Gfi-1 plays a central role in the phenotype of memory CD4 and CD8 cells induced by viral infection. More recently, it has been shown that Gfi-1 represses differentiation to the IL-17 and T%reg fates. Indeed, Gfi-1 appears to function as a repressor of fates alternate to Th2. In addition, an in depth effort to understand the distinctive signaling mechanisms used for IL-4 and IL-13 responses, with emphasis on the differential roles of the type I and type II IL-4 receptors and how different cell types show very different relative sensitivity to these congeners. For this purpose, responses of various macrophage populations from wild-type and gamma common-deficient mice to IL-4 and IL-13 have been analyzed in detail. Both the phosphorylation of STAT6, as measured by glow cytometric analysis, and the induction of arginase-1, a gene that is rapidly induced through the IL-4 receptor, has been evaluated. Bone marrow derived macrophage (BMDM) populations and monocytes are exquisitely sensitive to IL-4 but require 100 to 1000 fold more IL-13 to achieve a comparable response. Paradoxically, BMDM from gamma common-deficient mice respond well to IL-13 but poorly to IL-4 and anti- gamma common antibody essentially abolishes responses of monocytes to IL-4. Peritoneal macrophages and fibroblasts respond well to both IL-4 and IL-13 and gamma common deletion has very little effect on either IL-4 or IL-13 responses. Using information regarding the numbers of receptor subunits and equilibrium constants for each interaction, a model has been derived to explain these results. From the analysis, it has been concluded that IL-4 plays a central role as an inducer of differentiation while IL-13 is a major effector molecule. Building on this work, mutants of IL-4 have been prepared that have higher or lower affinity for gamma common or IL-13Ra, leading to predictable changes in the sensitivity of various cell types to IL-4 and have lead the prediction to a strategy to produce a super IL-4 that has enhanced signaling through the type I IL-4 receptor and reduced signaling through the type II IL-4 receptor. LI scientists have also developed new insights into the cytokine-stimulated production of IL-13 by basophils and mast cells. IL-3 activating STAT5 collaborates with IL-33 to activate mast cells IL-13 production;by contrast basophil IL-13 production can be stimulated most efficeintly by IL-3 plus IL-18. Laboratory scientists have developed a series of indicator mice. Particularly valuable are those that reflect the expression of IL-4, IL-13 and TSLP. These indicator mice were made through the introduction of a bacterial artificial chromosomes in which the AM-Cyan gene reported IL-4, destabilized DS-Red has reported IL-13 and ZS-green reported TSLP expression. These mice have proved to be excellent reporters of cytokine production in Schistosomiasis infection and their value in other infections is being studied.